1. Field of the Invention
The present invention relates to nucleotide sequences coding for the bovine .beta..sub.3 -adrenergic receptor (AR.beta..sub.3), to the use of the said sequences as probes and for the expression of peptides and/or of fragments of the latter having bovine AR.beta..sub.3 activity, to the vector which is useful for the said expression and also to host cells containing the said vector.
The present invention also relates to a method for the screening of substances possessing an agonist or antagonist action with respect to peptides of bovine origin having .beta..sub.3 -adrenergic receptor activity.
2. Description of the Related Art
Catecholamines such as adrenaline and noradrenaline, synthetic agonists of these catecholamines which mimic their biological functions and antagonists which block these biological functions are known to exert their effects by binding to specific recognition sites (membrane receptors) located on the cell membranes.
Two main classes of adrenergic receptors have been defined, .alpha.-adrenergic receptors and .beta.-adrenergic receptors.
In the set of these two classes, five subtypes of catecholamine receptors are now distinguished (.alpha..sub.1 -, .alpha..sub.2 -, .beta..sub.1 -, .beta..sub.2 - and .beta..sub.3 -AR). Their genes have recently been isolated and identified (S. COTECCHIA et al., 1988, Proc. Natl. Acad. Sci. USA, 85, 7159-7163; B. K. KOBILKA et al., 1987, Science, 238, 650-656; T. FRIELLE et al., 1987, Proc. Natl. Acad. Sci. USA 84, 7920-7924; L. J. EMORINE et al., 1987, Proc. Natl. Acad. Sci., USA, 84, 6995-6999; L. J. EMORINE et al., 1989, Science, 245, 1118-1121). Analysis of these genes has enabled them to be recognized as belonging to a family of integral membrane receptors displaying certain homologies (R. A. F. DIXON et al., 1988, Annual Reports in Medicinal Chemistry, 221-233; L. J. EMORINE et al., 1988, Proc. NATO Adv. Res. Workshop), in particular in respect of 7 transmembrane regions which are coupled to regulatory proteins, known as G proteins, capable of binding guanosine triphosphate (GTP) molecules.
These membrane receptors, when they have bound the appropriate ligand (agonist or antagonist), undergo a change in conformation, which induces an intra-cellular signal which modifies the behaviour of the target cell.
In the case of .beta.-adrenergic receptors, when they bind to catecholamine agonists, they catalyse the activation of a class of G proteins, which in turn stimulates adenylate cyclase activity, while AR.beta. antagonists act in competition with the agonists for binding to the receptor and prevent activation of adenylate cyclase.
When adenylate cyclase is activated, it catalyses the production of an intracellular mediator or second messenger, in particular cyclic AMP.
The inventors have recently demonstrated new .beta.-adrenergic receptors in man, designated Hu-AR.beta..sub.3, and in the mouse (International Application WO 92/12,246), designated Mu-AR.beta..sub.3, and characterized by properties different from those of the .beta..sub.1 and .beta..sub.2 receptors, in particular in that they behave differently with respect to substances which are, respectively, .beta..sub.1 - and .beta..sub.2 -receptor antagonists and agonists (International Application WO 90/08,775).
In particular, the Hu-AR.beta..sub.3 receptor consists, more especially, of a sequence of 408 amino acids, and is considered to contain seven hydrophobic transmembrane regions separated by intra- and extracellular hydrophilic loops, and the Mu-AR.beta..sub.3 receptor consists of a sequence of 400 amino acids and also contains 7 transmembrane regions.
The previous studies relating to Hu-AR.beta..sub.3 and Mu-AR.beta..sub.3 showed, in particular, that the .beta..sub.3 -adrenergic receptor participates in disorders such as diabetes and/or obesity, in as much as it is expressed in tissues which play an important part in metabolism (adipose tissues, skeletal muscles in particular).
Continuing his studies along these lines, one of the inventors sought to demonstrate such a .beta..sub.3 -adrenergic receptor in cattle (Bo-AR.beta..sub.3), so as to be able to have available a tool for regulating the amount of fats in these animals, in particular with the object of improving the quality of the meat.